Use medium if possible, but if your compound is not water soluble, then DMSO will be your best bet. Try to keep the concentration below 0.2%. DMSO has its own biological activities at higher concentrations and can either kill your cells or protect them by acting as an antioxidant. Just include a DMSO control in your assay, and if you're doing a colorimetric assay (e.g., MTT) make sure your compound doesn't reduce MTT (some do), and if it's FACS based (Annexin V/PI), make sure your compound doesn't cause a shift on its on (most I've studied do).

Yes, I agree. Also, if you use alcohol or any other solvetn, you must check the maximum concentration of the solvent allowed by your cells. For example, if you use DMSO, the final concentration of DMSO should be below 0.5% (v:v).

You should also check the effect of this extract on cell adhesion and do not confuse it with apoptosis or necrosis since a lot of plant extracts detache cells.....

You disolve first your plant extract in DMSO or ethanol at high concentration (mM) which could be then stocked at -20°C. For working solutions, you can dilute your stock solution in cell culture medium and then filter it to sterilize. From this solution you may prepare sequential dilutions in the same cell culture medium (no need to filter again). These solutions might stocked at 4°C for sevaral days. Remebmer that your control culture without plant extract should contain DMSO or ethanol in the same proportion than your treated cells (0.5% or others).

You can use DMSO or alcohol for your plant extract. Usually maximum final concentration used for carrier < or = to 0.05% (v:v). Second, plate down your confluence cells to a certain concentration in each well (using well plate) and let the cells attached to the surface of well plate for overnight and start the treatment. Good Luck

Ideally, you first have to measure the density of the compound by measuring the mass of a known volume (for instance measuring the mass of 100micro litres). Then obtain 20mg of the extract and dissolve it in DMSO, which aids the miscibility especially if the extract is hydrophobic. Then dissolve the 20mg and make up a solution of 500microlitres. This will make up a stock solution of 40ug/uL. From the stock solution, you should do serial dilutions using your cell medium (e.g. RPMI or DMEM), for instance at 1ppm, 5ppm, 10ppm, 50ppm, 100ppm etc..,

Good luck with your work

It is better to add the extract at the time of log phase of the cell culture i.e. before it reach confluence state.

The plant extract is ideally dissolved in an inert solvent like DMSO to a final concentration like 20mg/L and filtered to remove any particulate matter. It is further serially diluted at the time of drug addition using the media used for culturing your cells. While working with microtitre plates,people usually give it 24 hours for the cells to get attached or acclimatized and then add the drug. For a flask, you can wait until it is 70-80% confluent. Hope this helps :)

I find that DMSO is one of the best vechicles to use to dilute most chemicals. But, when I treated with plant extracts, I just used the media the cells were growing in to do the dilutions. Just take into account any pH changes that the plant extracts might make. If you have media with phenol red you should be able to see that right away. You may have to adjust for that.

I also routinely treat cells slightly before confluence.

As mentioned before you can use DMSO or Ethanol for solving your extracts. We always go for 1:1000 dilutions (so 0.1% v/v).

When treating your cells for the very first time you may have a look on your cells before and after treatment to make sure that they can deal with it ;). Depending on the sensitivity of your cells you may need longer 'attachment times' (I have one cell line which I culture 48h after seeding and then start treatment). However, seeding cells 24 h before treatment is a good approach and usually works out. Also we experienced that it is not necessary to filter or sterilize the solved substances or extracts before using them for cell culture (both solvents are cell toxic in high concentrations) (BUT : the cell culture medium always contains Pen/Strep).

Another point: do you know if your plant extract might be metabolized or degraded? So I wouldn't keep it dissolved at low concentrations at or above 4°C. Same is true for the cell culture, so if you would like to treat your cells longer than 24 h a 'refreshing' of the medium including your plant extracts might be necessary.

Have fun

Many articles suggest that you should use DMSO instead of ethanol but I recently did cytotoxicity assay with ethanol and DMSO because I have to do the same, so I got 1% cell death in ethanol as well as in DMSO by taking 10 ul with 90 ul of DMEM + FBS. Even in one article, they were using 0.01% of DMSO v.v. so, its better for you to dissolve that plant extract in DMSO and then dilute it, even u also can try MTT assay to check whether DMSO is giving more killing or Ethanol and then u can use DMSO or ethanol and do it in ur own way!! and after confluency (70%), you should add..

In my lab we work with multiple extracts all the time. We try to dissolve the extract in the media. DMSO, adds some complications, but can be used if a highly concentrate stock is made with the extracts. We typically treat cells at 50% + confluency, depending on the experiment. Feel free to email me if you want to discuss your protocol in more detail. Also you can check out one of our manuscripts (Cagle, et al 2012, rosehip extracts in brain tumor cells).

I would say, would be best to add it when it is 70-75% confluent

the drug/extract should be dissolved in culture media w/o serum for the treatment

If required, u can use dmso to increase the solubility

Your question needs more clarification. Do you need to add plant extract as a powder or as a solution. It depends on your preparation or extraction method. If your extraction was alcoholic , you need to get rid of any alcohol residuals then you can can make serial dilutions from your extract to assess its effect. If you extraction was watery , you can just add it according to your desire concentration.

Thank you for your invaluable input. I will get back to you with an update of the preliminary results. @Nahi Yaseen i will add the plant extract in solution form.

I can’t copy the answer of Nahi Yaseen. However my answer is the same..

DMSO can be differentiating agent or citotoxyc citocyde (depending on final concentration in the medium). DMSO can interfere with adhesion to flash (if you use adherent cells).

Not all tumoral cells are equal, their behaviour can be different.

Your extract il liquid? Is water-soluble?

That is fine, if your extracy is in a liquid form you can follow Dixon concept to make yiur dilutions and to assess yiur extarct impact.

For cude extracts as in your case, you can dissolve/dilute the extracts in the medium in which it was prepared (eg. 50% EtOH or 80% MeOH), vortex, centrifuge and filter sterilize You . DMSO is equally suitable and highly recommended for pure compounds. On the other hand, you can dilute extracts in media used for cell growth. Its also advisable to check the effect of the media being used to dissolve/dilute the extracts on the cell.

Extracts should be added when cells are confluent enough and incubated (eg. for 24hrs) to know the morphological & other effects caused by the extract.

Do keep us informed on the progress of your study.

Regards!

Use medium if possible, but if your compound is not water soluble, then DMSO will be your best bet. Try to keep the concentration below 0.2%. DMSO has its own biological activities at higher concentrations and can either kill your cells or protect them by acting as an antioxidant. Just include a DMSO control in your assay, and if you're doing a colorimetric assay (e.g., MTT) make sure your compound doesn't reduce MTT (some do), and if it's FACS based (Annexin V/PI), make sure your compound doesn't cause a shift on its on (most I've studied do).

@Ashley Hilchie....if i use the compound alone, how will i ensure that the solution is sterile. I have read in articles that DMSO (1% i.e ratio of 1:99) assist to make the medium sterile.

Filter sterilization

Prepare your stocks in DMSO and dilute in medium. You can then filter sterilize this mixture if you wish. Alternatively, you can just use it as is (I would do this since filter sterilization may alter the concentration of your compound). I have never had an issue with contamination of my stocks that were prepared in DMSO (provided that the medium is sterile), so I never filtered them.

Also, your product will likely be stable in DMSO for long term storage. We prepared our stocks (100 mM) in DMSO and stored them at -80. Our highest working concentration was 100 uM and they were still biologically active after a year or so.

You need to prepare your plant extract 100x conc in that case your DMSO/Alchol concentration will be below 0.01%